Inhibitors of HIV-induced downregulation of HLA class I Multiple studies suggest that downmodulation of HLA class I (MHC-I) plays an important role in HIV infection. The viral protein Nef induces MHC-I downmodulation and interferes with the presentation of viral antigens, making HIV-infected cells less sensitive to killing by CD8-positive CTLs. Macaques infected with SIV deficient in this activity rapidly restore the ability to eliminate MHC-I. The mechanism by which Nef downmodulates MHC-I has been extensively investigated, however, despite its important role in pathogenesis this activity remains to be targeted for antiviral development. Further highlighting the importance of this function in immune evasion, interference with antigen presentation has also been recognized in many herpesviruses, poxviruses, papillomaviruses and adenoviruses. We propose screening a library of small molecules to identify inhibitors of the HIV-induced MHC-I downmodulation. A high content screen has been fully optimized and preliminary findings from a test pilot of 3,440 small-molecules have validated the assay and identified small-molecules blocking this promising target. Our results demonstrate proof-of-concept that potent and specific interference of MHC-I downmodulation with small molecules is possible. We propose the following aims: (1) to screen a library of 46,280 drug-like molecules composed of highly diverse structures, including molecules selected to target kinases, and other compounds known to be pharmacologically active (Lopac library); (2) to validate and characterize the potency, specificity and toxicity of hts, and; (3) to identify compounds that enhance the ability of CTLs to kill HIV- infected cells. We wil use a multi-modal HTS assay with multiple built-in controls to simultaneously confirm hits in primary screens and eliminate false positives with poor toxicity profiles, and other hits blocking HIV infection by known mechanisms. Success in this Phase I application will be defined by identifying one or more bona fide lead series of drug-like compounds with EC50s below 5M, minimal cytotoxicity (CC50 at least 30-fold greater than EC50), and the ability to sensitize HIV-infected cells to CTL killing. In a Phase II proposal, chemical leads from this screen will be selected for medicinal chemistry SAR optimization for potency, specificity and oral bioavailability. We wil further study the mechanism of action, characterize drug-resistant strains, ases the potential for antagonism with approved antiretrovirals, and evaluate cross-resistance with other drugs. Additionally, a selected number of compounds will be tested in a small-animal model of HIV infection that recapitulates the virus-specific CTL responses. Compounds with improved profiles and characterized mechanism of action will be advanced into preclinical development studies to support future human trials. Administration of antivirals inhibiting MHC-I downmodulation may lengthen the time required before initiation of HAART therapy, or alternatively, in combination with other drugs may enhance their antiviral effect by improving the patient's immune response against the virus. These studies will constitute the foundation of a drug discovery program that may lead to first-in-class drugs of a novel family of antivirals with the potential for broader activity against other viruses. PHS 398/2590 (Rev. 06/09) Page Continuation Format Page